Formation of nascent DNA molecules during inhibition of replicon initiation in mammalian cells.

X-irradiation of mammalian cells with moderate doses (100-1000 rads) inhibits the initiation of DNA replicons. This inhibition is observed as depressed amounts of radioactivity at low molecular weights when the DNA from the cells is analysed by velocity sedimentation in alkaline sucrose gradients at 30 min after irradiation. There is no detectable effect on chain elongation and joining of those molecules that do initiate replication; this is indicated by the presence of the same amounts of radioactivity in nascent DNA molecules of high molecular weights from control and irradiated cells. The labeling of DNA molecules that initiated replication before irradiation continues unhindered for more than 60 min after irradiation, which is observed as peaks of radioactivity at high S values in alkaline sucrose gradients from irradiated cells. These data indicate that DNA replication in mammalian cells proceeds by continuous joining of nascent molecules that initiate almost simultaneously at origins at various distances from one another. Some of the interorigin distances are much greater than others, implying that large replicons make up a significant component of mammalian DNA.     

Excess thymidine accelerates nascent replicon maturation without affecting the rate of DNA synthesis

The effects of different concentrations of exogenously supplied dThd on DNA replication were investigated in seedlings of Pisum sativum. Nascent DNA was labeled with either [³H]dThd or [³H]dAdo in the presence of 1·10^?6, 1·10^?5 or 1·10^?4 M unlabeled dThd. The rate of DNA synthesis was determined by measuring the kinetics of radioactivity incorporation into trichloroacetic acid-precipitable material and the size of the nascent molecules was investigated using alkaline sucrose gradients. The results obtained showed that high concentrations of exogenously supplied dThd accelerated the joining of completed nascent replicons without affecting the rate of DNA synthesis. This observation strengthens the hypothesis that the dTTP pool size is one of the factors controlling the timing of nascent replicon maturation.     

In the higher plant Pisum sativum maturation of nascent DNA is blocked by cycloheximide, but only after 4-8 replicons are joined.

Velocity sedimentation in alkaline sucrose gradients, single cell autoradiography and cytophotometry were used to determine if protein synthesis is required for the maturation of nascent replicons to chromosomal-sized molecules in cultured pea-root cells. The results obtained showed that cycloheximide at 5 and 10 microgram/ml, added either before or during labeling with tritiated thymidine, blocked maturation of nascent DNA at an intermediate size of 72-140 X 10(6) daltons single-stranded DNA. To reach this size, nascent replicons - which are 18 X 10(6) daltons single-stranded DNA each - were replicated and groups of 4-8 replicons were joined even though protein synthesis was reduced to 15% of the control. Further maturation of the nascent molecules to chromosomal size, however, was prevented and this resulted in the accumulation of nascent molecules in the 72-140 X 10(6) daltons range. The experiments also showed that the joining of nascent replicons is not an absolute function of late S or G2 phase of the cell cycle, since cells treated with cycloheximide and blocked in mid-S phase had nascent DNA of a size corresponding to 4-8 joined replicons. Finally, the results support the hypothesis that at least one step in the process of nascent DNA maturation may require replication, during late-S phase, of DNA segments that are interspersed within replicon-clusters that replicate early in the S phase.     

Synthesis of high molecular weight DNA strands during S phase

The organization of the mammalian S phase was studied in synchronized mouse embryo cells in terms of the spatial relationship between replication units whose synthesis is initiated at different times in S phase and the rate of assimilation of replication units into high molecular weight DNA strands.The formation of high molecular weight nascent DNA strands several replication units in length was analyzed by velocity sedimentation in alkaline sucrose gradients and by isopycnic centrifugation in alkaline Cs₂SO₄/CsCl gradients. Differential labeling with an isotopic and a density label shows that replication units synthesized at different stages of the S phase are not found within the same high molecular weight polynucleotide strand. It is thus concluded that replication units duplicated at different stages of the S phase are spatially organized in clusters along the mammalian genome.The rate of formation of high molecular weight nascent DNA strands is at least 4 to 8 times slower than that predicted from the spatial organization of replication units and the rate of chain growth within replication units. It is concluded that the process of joining of the completed nascent strands of adjacent replication units plays a major role in the rate of completion of high molecular weight strands.     

DNA replication in Physarum polycephalum: bidirectional replication of DNA within replicons.

The direction of replication of DNA within replicons of Physarum polycephalum was studied by pulse-labelling with 5-bromouracil-deoxyriboside (BrdUrd) and 3H-adenosine deoxyriboside (dAdo), followed by ultraviolet- (UV) -photolysis and analysis of molecular weights of single strand DNA fragments on alkaline sucrose gradients. Newly made DNA within replicons at all stages of completion is split in two equal halves upon UV irradiation when BrdUrd was given at the time of initiation of DNA synthesis. This shows that replication within replicons of Physarum polycephalum starts at an origin located in the center of each unit, proceeding bidirectionally from this origin.     

Nuclear DNA synthesis is blocked by UV irradiation in Dictyostelium discoideum

We have used a thymidine auxotroph of the simple eukaryote, Dictyostelium discoideum and alkaline sucrose gradients of isolated nuclei to study alterations in DNA synthesis following irradiation of replicating haploid cells with 254 nm UV light. Three responses were characterized using pulse-chase protocols: (1) Lags in DNA synthesis as measured by the amount of label incorporated were 4, 9, and 20 h after 10, 50, and 200 J/m2. (2) The DNA synthesized during a 15-min pulse immediately after irradiation was of lower single strand molecular weight: 7, 3.5, and 3 x 10(6) dalton after 0, 50, and 200 J/m2. (3) The time required for maturation of the nascent DNA to full-sized single strands of about 2 x 10(8) dalton was 45-50 min for unirradiated cells, 3 h after 10 J/m2, and 20 h after 200 J/m2. The DNA of the irradiated cells did not mature uniformly during these delays; instead, a period of no increase in size was followed by a rapid, nearly control rate of maturation. We conclude: (a) at least some UV lesions block elongation of replicons; (b) the elongation of the replicons and their subsequent joining to yield mature high molecular weight DNA occurs after most of the lesions are repaired; (c) the timing of the different aspects of recovery suggest that initiation of replication is also inhibited.     

Changes in structural integrity of heart DNA from aging mice

The state of the structural integrity of the DNA from mouse myocardial cells has been investigated by utilizing both CsCl density gradient sedimentation and digestion by S₁ endonuclease from $\text{Aspergillus}$$\text{orzae}$. The DNA from myocardial cells of young mice sedimented in a narrow peak at the expected density of 1.701 g/cm³, while the DNA from the heart cells of senescent mice became broadly distributed in CsCl gradients, banding even more multimodally in alkaline sucrose gradients. This mode of sedimentation indicates that old mouse DNA becomes partially fragmented. When the native DNA of myocardial cells from 6, 20 and 30 month old mice was treated with single-strand specific S₁ endonuclease, it was the DNA from the senescent mice that showed a progressive increase in sensitivity to digestion by the enzyme. The results indicate that the heart DNA of aging mice develops single-stranded gaps in addition to a breakdown into differently sized fragments.     

The effects of different thymidine concentrations on DNA replication in pea-root cells synchronized by a protracted 5-fluorodeoxyuridine treatment

Single-cell and DNA fiber autoradiography, cytophotometry and velocity sedimentation in alkaline sucrose gradients were used to analyse DNA replication and nascent replicon maturation in 5-fluorodeoxyuridine (FUdR)-synchronized cells of Pisum sativum. The replicon size was not significantly changed by the protracted FUdR treatment. When the synchronized cells were released from the inhibitor, labeled with [³H]TdR for 30 min, and chased in medium containing 1 × 10⁻⁶ M or lower concentrations of cold thymidine, DNA replication stopped after approx. 25% of the genome had replicated, and the nascent strands failed to grow above 9–12 × 10⁶ D single-stranded (ss) DNA. When the cells were chased in medium with 1 × 10⁻⁵ M cold thymidine, the DNA content of the labeled cells steadily increased with time and the size of the nascent molecules grew continuously until replicon size was achieved; then they were accumulated at replicon size until the cells arrived in late S or G2. When the FUdR-synchronized cells were chased in medium containing 1 × 10⁻⁴ M cold thymidine, the size of the nascent strands increased continuously with time, indicating that some neighbouring nascent replicons were joined as soon as they completed their replication. These observations led us to postulate that in FUdR-synchronized cells the rates of chain elongation, cell progression through the S phase and nascent replicon maturation are controlled by thymidine availability.     

DNA replication in Physarum polycephalum: UV photolysis of maturing 5-bromo-deoxyuridine substituted DNA.

Combinations of 5-bromodeoxyuridine (BrdUrd) and 3H-deoxyadenosine (3H-DAdo) short pulses were given in the synchronous DNA-replication period of Physarum polycephalum. After a chase period, UV-photolysis products were analyzed on alkaline sucrose gradients. This strategy has allowed the following conclusions. a) at the time of master-initiation of DNA replication, points separated by 1.1-2.2x10(7) daltons of single strand DNA may initiate DNA synthesis. b) among these, only selected groups of replicons actually proceed in DNA replication at this time, while others appear to hold (later temporal sets of replicons). The origins of the ones that proceed in replication are separated from each other by a distance corresponding to 1.1-2.x10(7) daltons. c) regions in actual replication are separated from each other by increasing distances (up to 1.5x10(8) daltons single strand DNA) at later times in S.     

Molecular characterization of a stable Flac plasmid

F$\text{lac}$S is a thermostable extrachromosomal element isolated in $\text{Salmonella typhimurium}$ which is altered in its replication as compared to its precursor F_ts114$\text{lac}$. Sedimentation of both these plasmids in alkaline sucrose gradients has indicated a difference in their sizes. Contour length measurements of open circular plasmid DNA molecules photographed in the electron microscope have revealed the estimated molecular weight of F_ts114$\text{lac}$ to be 81 × 10⁶ daltons while that of F$\text{lac}$S is 109 × 10⁶ daltons. F$\text{lac}$S may carry a segment of $\text{S. typhimurium}$ chromosomal or cryptic plasmid DNA.     

Size distribution and maturation of newly replicated DNA through the S and G2 phases of physarum polycephalum

The size distribution of newly made DNA and the dynamics of size maturation of progeny DNA molecules were studied in the synchronous S and G2 phases of Physarum polycephalum. Pulse labeling of DNA and analysis of the products on alkaline sucrose gradients showed that synthesis of primary replication units (which will also be referred to as “Okazaki” fragments) occurred throughout the S period. Pulse and pulse-chase experiments revealed a distinct pattern of size maturation. An apparently linear increase in molecular weight of progeny DNA molecules during the first hour of the S phase occurred at a rate of approximately 4–5 × 10⁵ daltons per min at 26°C, corresponding to the joining of 6–8 Okazaki fragments. The resulting 35–45S (1.1–2.2 × 10⁷ daltons) DNA molecules may correspond to the Physarum “replicon.” The further size increases of the newly made DNA appear to occur in steps, possibly reflecting a clustering of isochronous replicons along the chromatide. These observations are discussed with regard to mechanisms of DNA replication and size maturation.     

Effect of cytosine arabinoside on replicon initiation in human lymphoblasts

Cytosine arabinoside inhibited DNA synthesis in human lymphoblasts by inhibiting the initiation of DNA replication units. This effect was observed by a decrease in the incorporation of (³H) thymidine into low molecular weight DNA analyzed by velocity sedimentation in alkaline sucrose gradients. In contrast, there was no detectable effect on chain elongation and joining of those molecules that initiated replication before addition of the drug. These data indicate that cytosine arabinoside acts preferentially at the level of initiation of DNA replication rather than chain elongation.     

Intermediate in adenovirus type 2 replication.

Replicating chromosomes, called intermediate DNA, have been extracted from the adenovirus replication complex. Compared to mature molecules, intermediate DNA had a greater buoyant density in CsCl gradients and ethidium bromide-cesium chloride gradients. Digestion of intermediate DNA with S1 endonuclease, but not with RNase, abolished the difference in densities. These properties suggest that replicating molecules contain extensive regions of parental single strands. Although intermediate DNA sedimented faster than marker viral DNA in neutral sucrose gradients, single strands longer than unit length could not be detected after alkaline denaturation. Integral size classes of nascent chains in intermediate DNA suggest a relationship between units of replication and the nucleoprotein structure of the virus chromosome. Adenovirus DNA was replicated at a rate of 0.7 x 10-6 daltons/min. Although newly synthesized molecules had the same sedimentation coefficient and buoyant density as mature chromosomes, they still contained single-strand interruptions. Complete joining of daughter strands required an additional 15 to 20 min.     

Caffeine enhancement of the initiation of DNA replication in benzo[a]pyrene diol epoxide damaged cells

We have used a newly developed pH stepwise alkaline elution method to show that caffeine enhances DNA initiation (DNA replication in sub-replicon size nascent strands) in (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene (BPDEI) damaged mouse primary epidermal cells. Caffeine alone caused a dose-dependent increase in DNA initiation without an effect on DNA elongation (joining of replicon-sized nascent DNA). BPDEI alone inhibited DNA elongation as shown by a relative increase in sub-replicon size nascent DNA. When BPDEI treated cells were incubated with caffeine, there was a dose-dependent increase in sub-replicon size nascent DNA without a significant effect on the proportion of joined replicons. Therefore, caffeine can enhance DNA initiation in mammalian cells damaged with a reactive form of the carcinogen benzo[a]pyrene and this may account for the biological interaction between caffeine and the ultimate carcinogenic form of benzo[a]pyrene.     

Replicon sizes in mammalian cells as estimated by an x-ray plus bromodeoxyuridine photolysis method.

A new method is described for estimating replicon sizes in mammalian cells. Cultures were pulse labeled with [3H]thymidine ([3H]TdR) and bromodeoxyuridine (BrdUrd) for up to 1 h. The lengths of the resulting labeled regions of DNA, Lobs, were estimated by a technique wherein the change in molecular weight of nascent DNA strands, induced by 313 nm light, is measured by velocity sedimentation in alkaline sucrose gradients. If cells are exposed to 1,000 rads of X-rays immediately before pulse labeling, initiation of replicon operation is blocked, although chain elongation proceeds almost normally. Under these conditions Lobs continues to increase only until operating replicons have completed their replication. This value for Lobs then remains constant as long as the block to initiation remains and represents an estimate for the average size of replicons operating in the cells before X-irradiation. For human diploid fibroblasts and human HeLa cells this estimated average size is approximately 17 micron, whereas for Chinese hamster ovary cells, the average replicon size is about 42 micron.     

DNA replication in Physarum polycephalum: characterization of replication products made in isolated nuclei

Nuclei isolated from synchronous S-phase plasmodia of the myxomycete $\text{Physarum polycephalum}$ were competent in production of low molecular weight DNA replication intermediates. Furthermore, these nuclei showed some competence in joining these fragments into DNA of intermediate molecular weight. The DNA molecules made $\text{in vitro}$ could be correlated with products made $\text{in vivo}$.     

Inhibitory effects of high doses of conA on S phase lymphocytes. ATP pool modification and rapid switch-off on new replicon initiations.

When S phase lymphocytes were treated for various times with high doses of ConA, we observed that labelled precursor incorporation into DNA was suppressed. This inhibition is characterized by its rapid onset, its lectin dose dependence and reversibility by α-methyl-mannoside. The uptake of labelled desoxyribonucleoside precursors is not modified by the treatment. The nucleoside kinase activity tested on cellular extracts showed a slight but significant decrease. However, the fact that the specific activity of newly replicated DNA was not modified indicates that the DNA labelling suppression is not a direct consequence of alterations in pathways of labelled DNA precursor synthesis. The available ATP pool in treated cells decreased by 25% after 30 min and near 50% after 1 h. The decrease in DNA labelling observed is related to a decrease in the overall rate of DNA synthesis. From density shift analysis of very large DNA molecules labelled by [${}^{125}\text{I]UdR}\text{BUdR}$ (a large part of a cluster of replicons), as well as velocity sedimentation analysis of pulse-chased molecules, we have demonstrated that (i) the DNA elongation within active replicons is not blocked; (ii) the rate of assembly of newly replicated DNA fragments (replicons) seems to be unmodified. Consequently, the initiations of adjacent replicons in operating clusters are not affected. However, the number of clusters which start their replication by initiation of new replicons is greatly reduced after 1 h of ConA treatment.     

Association of R6K plasmid replicative intermediates with the folded chromosome of Escherichiacoli

When $\text{Escherichia}\text{coli}$ strain CSH50(R6K) is lysed so as to preserve the folded chromosome structure approximately 9 of the 11 R6K molecules maintained per chromosomal equivalent cosediment with the host nucleoid on a neutral sucrose gradient; the remaining 2 plasmids sediment at their normal rate. When cells are briefly labeled with [³H]thymidine, the majority of plasmid replicative intermediates and nascent mature plasmids are found in the plasmid subpopulation that cosediments with host folded chromosomes. This finding suggests that plasmid replication occurs in a restricted cellular locus, perhaps even while in association with its host's folded chromosome.